Upon pathogen exposure, the innate immune system initiates a cytokine response such that the adaptive immune system is tailored to eradicate that pathogen (4, 11, 24). Defects in the innate immune signaling pathways result in patient susceptibility to infectious disease (4, 11, 24, 26). IRAK4 (IL-1 Receptor Associated Kinase 4) is a key signaling molecule that links extracellular pathogen exposure to cytokine transcription and release, and recently, a series of unrelated children who develop recurrent pyogenic infections have been found to have mutations in the IRAK4 gene (6, 15, 16, 22, 25). The mutations in IRAK4 cause C-terminal truncations and result in either the absence of the kinase domain of the molecule or the absence of the total IRAK4 protein (6, 15, 16, 22, 25). IRAK4 is part of a signaling pathway linking the Toll-like Receptors to the NF?B pathway and the MAP Kinase pathways. Mice genetically deleted for IRAK4 show an inability to respond to a variety of pathogen-associated molecules including LPS, peptidoglycan, viral RNA and bacterial DNA. In addition, while showing a normal TNF response, these mice cannot respond to IL-1 (30-33). Surprisingly, patients with mutated IRAK4 show a subtly different phenotype than that found in the mouse as these patients are predominantly susceptible to recurrent gram-positive pyogenic bacterial infections (6, 15, 16, 22, 25, 26). While IRAK4's kinase activity plays a clear role in these children's immunodefiency, the role of IRAK4's kinase activity in TLR signaling is unclear (13, 14, 18, 19, 27). Conflicting data has been published on the requirement of IRAK4 kinase activity for TLR responses and IRAK4's in vivo substrates and kinetics of activation are unknown. Given the importance of IRAK4 in mediating innate immunity, and given the fact that children with immunodeficiency caused by an IRAK4 mutation all show a deletion in the kinase domain, understanding the kinetics, spatial localization, and specificity of IRAK4's kinase activity will be important. It will also be important to determine the features that cause differences in IRAK4's role in gram-positive and gram-negative infections. The central hypothesis of this grant application is that IRAK4's kinase activity is responsible for effective signaling responses to gram-positive organisms and that differences in this kinase activity (in regards to activity and spatial localization) may underlie the infectious pathology in children with IRAK4 deficiencies. This grant aims to develop a novel in vivo signaling reporter such that IRAK4's kinase activity can be studied in vivo. If successful, this pilot grant will lead to insights regarding IRAK4's role in immunodeficiencies and will generate reagents that will help carry the work forward in animal models. PUBLIC HEALTH RELEVANCE: People with immunodeficiency syndromes are highly susceptible to infection with bacteria, fungi and viruses. They do not respond normally to these agents, and their immune systems cannot defend them against these pathogens. A mutation in a gene called IRAK4 gives rise to a newly discovered immunodeficiency syndrome. Patients with mutations in IRAK4 develop numerous recurrent infections to fever-causing bacteria. 43% of these patients die in childhood. It is important to understand the function of IRAK4 so that we can better understand the cause of this immunodeficiency syndrome and ultimately, better treat this immunodeficiency syndrome. This grant aims to develop novel reagents with which to study IRAK4 so that this immunodeficiency syndrome can be better understood.